Arteriosclerosis, Vol 7, 627-636, Copyright © 1987 by American Heart Association

ARTICLES

Modulation of glycosaminoglycan production and antithrombin III binding by cultured aortic endothelial cells treated with 4-methylumbelliferyl- beta-D-xyloside

K Shimada and T Ozawa
Department of Medicine and Geriatrics, Kochi Medical School, Japan.

The interaction between antithrombin III and heparinlike glycosaminoglycan molecules present on the vascular surface seems to play a significant role in the regulation of coagulation. We have tested the hypothesis that altered synthesis of glycosaminoglycans by endothelial cells could influence this interaction by using 4- methylumbelliferyl-beta-D-xyloside for metabolic perturbation of glycosaminoglycan production. Incubation of purified porcine 125I- antithrombin III with cultured porcine aortic endothelial cells demonstrated specific, time-dependent, saturable binding of this protease inhibitor to the endothelial cell surface with antithrombin III concentration at half-maximal binding of approximately 40 nM. This binding was displaced by heparin and was completely abolished by selective removal of heparan sulfate from cells with heparitinase, indicating that antithrombin III binds to heparan sulfate on the surface of endothelial cells. Incubation of cell cultures with beta-D- xyloside resulted in a reduction of maximum antithrombin III binding by approximately 65% with little alteration in binding affinity. beta-D- Xyloside did not affect the cellular growth or morphology. Reduction of the binding after exposure to various concentrations (33 to 500 microM) of xyloside occurred in parallel with the decrease in incorporation of both 35S-sulfate and 3H-glucosamine into cell surface heparan sulfate. Whereas the size of heparan sulfate chains derived from the cell surface was not altered by xyloside treatment, they appeared to have slightly less net negative charge and a significantly reduced proportion of the molecule with high affinity for antithrombin III in the presence of xyloside. On the other hand, secretion of free chondroitin sulfate (and dermatan sulfate) chains into the medium was markedly increased (16-fold) in the presence of xyloside, accompanied by a smaller increase in secretion of free heparan sulfate chains. There was a good correlation between conditions with decreased antithrombin III binding and an inhibition of the endothelial cell- mediated acceleration of thrombin inactivation by antithrombin III. These results suggest that beta-D-xyloside caused a dose-dependent decrease in production as well as some subtle structural alterations of cell-surface-associated heparan sulfate, which could serve as binding sites for antithrombin III on the endothelial cells and mediate enhancing the anticoagulant activity of this protein. This system may offer a potentially useful model to investigate the possible mechanisms responsible for the development of a procoagulant state involving these endothelial macromolecules.

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